Neurotransmitters are critical to the regulation of the central and peripheral nervous systems and command a number of functions such as learning, memory, sleep, and movement. Deng et al., Determination of amino acid neurotransmitters in human cerebrospinal fluid and saliva by capillary electrophoresis with laser-induced fluorescence detection, J. Sep. Sci. 2008, 31, 3088-3097; Boyd et al. Trace-level amino acid analysis by capillary liquid chromatography and application to in vivo microdialysis sampling with 10-s temporal resolution, Anal. Chem. 2000, 72, 865-871. Discerning the machinery involved in vesicular fusion, the spatiotemporal mechanisms of synaptic release, and the chemical activity of neurotransmitters is vital to understanding both normal and atypical cellular processes. The ability to effectively monitor exocytotic operations bolsters research in neuroscience, serving as a useful tool in the study of neurophysiology and neuropsychiatric disorders. Methods to evaluate exocytosis include fluorescence imaging, capillary electrophoresis, microelectrochemistry, and mass spectrometry. Gubernator et al., Fluorescent false neurotransmitters visualize dopamine release from individual presynaptic terminals, Science 2009, 324, 1441-1444; Steyer et al. Transport, docking and exocytosis of single secretory granules in live chromaffin cells, Nature 1997, 388, 474-478; Miesenböck et al., Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins, Nature 1998, 394, 192-195; Felmy, Modulation of cargo release from dense core granules by size and actin network, Traffic 2007, 8, 983-997; Burchfield et al., Exocytotic vesicle behavior assessed by total internal reflection fluorescence microscopy, Traffic 2010, 11, 429-439; Kennedy et al., Microcolumn separations and the analysis of single cells, Science 1989, 246, 57-63; Kristensen et al., Capillary electrophoresis of single cells: observation of two compartments of neurotransmitter vesicles, J. Neurosci. Meth. 1994, 51, 183-188; Chang et al., Determination of catecholamines in single adrenal medullary cells by capillary electrophoresis and laser-induced native fluorescence, Anal. Chem. 1995, 67, 1079-1083; Omiatek et al., Only a fraction of quantal content is released during exocytosis as revealed by electrochemical cytometry of secretory vesicles, ACS Chem. Neurosci. 2010, 1, 234-245; Ponchon et al., Normal pulse polarography with carbon fiber electrodes for in vitro and in vivo determination of catecholamines, Anal. Chem. 1979, 51, 1483-1486; Leszcezyszyn et al., Nicotinic receptor-mediated catecholamine secretion from individual chromaffin cells, Chemical evidence for exocytosis, J. Biol. Chem. 1990, 265, 14736-14737; Li et al., Single-cell MALDI: a new tool for direct peptide profiling, Trends Biotechnol. 2000, 18, 151-160. Non-optical techniques are limited by poor throughput and a lack of spatial resolution. Ge et al., Bioanalytical tools for single-cell study of exocytosis, Anal. Bioanal. Chem. 2010, 397, 3281-3304. Conversely, fluorescence methods offer a sensitive means to elucidate the spatial distribution of neuronal vesicles and chemical messengers.
Fluorescence imaging of secretion was studied early on by loading chromaffin cells with acridine orange and observing a loss in fluorescence upon exocytosis. Steyer et al., Transport, docking and exocytosis of single secretory granules in live chromaffin cells, Nature 1997, 388, 474-478. More recently, exocytosis has been visualized using the genetically-encoded synapto-pHluorins, wherein a pH-sensitive GFP construct is expressed on the inner membrane of secretory vesicles. The engineered vesicles fluoresce upon exocytosis due to a change in pH from the acidic synaptic vesicle (˜5) to the neutral synaptic cleft (˜7.4). Miesenböck et al., Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins, Nature 1998, 394, 192-195. These methods solely monitor the process of vesicle membrane fusion during an exocytotic event but do not directly image active neurotransmitters released upon exocytosis. In recent years, a genetically-encoded CFP/YFP FRET biosensor was developed to monitor glutamate release, spillover, and reuptake by fluorescence. Hires et al., Optical measurement of synaptic glutamate spillover and reuptake by linker optimized glutamate-sensitive fluorescent reporters, P. Natl. Acad. Sci. 2008, 105, 4411-4416. However, these protein-based biosensors require genetic manipulation and display high, irreversible affinity for glutamate with limited dynamic range and overall small changes in fluorescence. As a result, these protein-based sensors are neither intended for, nor compatible with, specialized neurosecretory cells (e.g., glutamatergic, dopaminergic, etc.) that possess high concentrations of primary amine neurotransmitters (300 mM-1M). To avoid the use of protein-based fluorophores, a pH sensitive fluorescent false neurotransmitter (FFN) has been developed to monitor exocytosis. More specifically, FFNs solely monitor vesicular membrane fusion with the cellular membrane. This fluorescent tracer is loaded into vesicles expressing VMAT and fluoresces upon exocytosis similar to the synapto-pHluorins. Rodriguez et al., Fluorescent dopamine tracer resolves individual dopaminergic synapses and their activity in the brain, P. Natl. Acad. Sci. 2013, 110, 870-875. FFNs neither directly detect neurotransmitters nor directly monitor exocytotic release of neurotransmitters. There remains, however, a scarcity of small molecular sensors that can directly detect and image neurotransmitters upon exocytosis.
In recent years, coumarin aldehyde fluorescent sensors, such as the ones disclosed in U.S. Pat. No. 7,977,120 and International Application No. PCT/US2014/31490, were developed. Both the '120 sensor and the 31490 sensor, with the exemplary structures shown in FIG. 1, are fluorescent sensors for the selective recognition and sensing of amines. The '120 sensor with a boronic acid recognition unit, unfortunately, can be quenched by the catechol group upon binding, and thus, in the case of dopamine and norepinephrine, operated in a turn-off mode. The 31490 sensor is a turn-on sensor for the selective labeling and imaging of the dopamine and norepinephrine inside secretory vesicles. More specifically, the 31490 sensor enters the vesicle and binds to the primary amine of the catecholamine, creating a positively charged iminium ion. Formation of the iminium ion also induces a bathochromic shift in absorbance that can be selectively excited at 488 nm allowing the neurotransmitter to be imaged directly, giving the signature punctate fluorescence. Nettie et al., Selective catecholamine recognition with NeuroSensor 521: a fluorescent sensor for the visualization of norepinephrine in fixed and live cells, ACS Chem. Neurosci. 2013, 4, 918-923. The charged complex cannot translocate across the vesicular membrane and becomes trapped, accumulating inside the vesicle and selectively labeling only primary amine neurotransmitters present in high concentrations (50 mM-1M) within an acidic environment (e.g., secretory vesicle).
Therefore, there is a need for new pH-sensitive fluorescent chemosensors that directly detect and image neurotransmitters released upon exocytosis.